Tumour Cytotoxic Agent and Methods Thereof

ABSTRACT

The present invention discloses a novel antitumour agent which provides an antitumour activity based on metabolites with reduced side effects. Moreover, the antitumour agent is prepared starting with a highly safe bacterium used in food production, which is a probiotic lactic acid bacterium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of co-pending ApplicationSer. No. 13/579,936, filed Aug. 19, 2012; which is a National StageApplication of International Application No. PCT/MY2010/000307, filedNov. 29, 2010.

The Sequence Listing for this application is labeled“SeqList-17Jul13.txt”, which was created on Jul. 17, 2013, and is 6 KB.The entire content is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The present invention relates to a use of metabolites obtained fromGram-positive bacteria such as Lactic acid bacteria as an anti-tumouragent. Indeed, the present invention also discloses methods for treatingand/or preventing tumour growth on human cells.

BACKGROUND OF INVENTION

Lactic acid bacteria that existed widely in the natural world aremicrobes of generating an organic acid by using a carbohydrate such as aglucose or lactose (von Wright, 2005). The Lactic acid bacterium hasbeen directly or indirectly used in food from a long time ago. As aresult of a research on an intestinal microflora of a man, it has beenreported that the main Lactic acid bacteria in the gastrointestinaltrack of a healthy man are Lactobacillus acidophilus, Lactobacilluscasei, Lactobacillus fermentum, Lactobacillus plantarum and so on. SuchLactic acid bacteria are resistant to gastric acid and bile (Balcazar,2007). Presently, the Lactic acid bacterium has been widely used as amicrobial agent or vital cell preparation of fermented goods and so onall over the world. There are many reports about the cytotoxic effect offermented milk or Lactic acid bacteria on tumour cells. For example, theadministration of Lactobacillus casei can prevent the development ofcolorectal cancer, with a daily intake of live L. casei suppressingatypia of colorectal tumours in 398 men and women who were free fromtumours and who had at least two colorectal tumours removed (Ishikawa etal., 2005). Lactic acid bacteria are the bacteria metabolizingcarbohydrate and producing lactic acid thereby. These bacteria belong tofacultative anaerobes or obligatory anaerobes which proliferate wellunder anaerobic conditions. A few commonly known genuses of Lactic acidbacteria are Streptococcus, Lactobacillus, Leuconostoc, Bifidobacteriaand Pediococcus. A Streptococcus genus microorganism is ahomofermentative bacterium that generates lactic acid by fermenting milkto suppress putrefying bacteria or pathogenic bacteria. A Lactobacillusgenus microorganism is a bacilliform, and a homo- or heterofermentativebacterium, which is widely seen in the fermentation of dairy products orvegetables. A Leuconostoc genus microorganism, a diplococcus, is aheterofermentative bacterium and mostly involved in fermentingvegetables. A Bifidobacteria genus microorganism is an obligatoryanaerobe which generates L(+) lactic acid useful for children's health,but it cannot survive under aerobic conditions (Holzapfel et al., 2001).

Many chemical compositions have been reported as an anti-tumour agent.However, these compositions not only destroy the tumour cells, but alsodestroy normal cells. Alternatively, surgical treatment is generallyperformed to remove tumour tissue. However, this could be difficult asthis method does not remove the tumour tissues thoroughly. It is saidthat the actual condition of any therapy provides side effects and doesnot fully acquire the best results. For example, chemotherapy has beensuccessfully used as neo-adjuvant, adjuvant and salvage strategies(Carlson et al., 2006). Chemotherapy targets rapidly dividing cancercells, with deleterious side effect to rapidly dividing normal cells.This results in the most common side effects of chemotherapy such asendothelial toxicity, immunosuppression, mucositis and alopecia(Mitchell, 2004; Mailloux et al., 2005). Moreover, the use ofconventional chemotherapeutic drugs has been proved to causechemoresistance in cancer cells (Zhivotovsky and Orrenius, 2009). It isalso used as concomitant treatment therapy with irradiation andbiological agents (American Cancer Society, 2009). Despite much moreadvance in chemotherapy to surgery and irradiation, cancer mortalityrate still ranks high among causes of mortality in many countries (WHO,2009a). It is therefore essential to develop novel chemotherapeuticswith greater efficacy while limited toxicity to normal cells.

The desire by consumers to use natural methods for health maintenancerather than long-term chemotherapeutics agents linked to theirexpectation that food becomes a source of prolonged well-being, supportsthe speculation that the probiotic market will expand rapidly. Resultsobtained from multidisciplinary research will probably essential for thepositioning of probiotic preparations as either a food, a foodsupplement or as pharmaceutical preparation (Mercenier et al., 2002).Bacteriocins are isolated from Lactic acid bacteria and they exhibitinhibitory effects against various pathogens in a manner similar toantibiotics. However, bacteriocins are distinguishable from antibioticsin terms of their synthesis, mode of action, toxicity and resistancemechanisms.

New antitumour compound are continually in demand, for the treatment ofcancer in man and the production of new anticancer compounds is animportant feature of developing antitumour agents for further studies.Equally important are novel strains of cultures used in the productionprocesses for preparing these compounds. Additionally, it has beendetermined that the metabolites produced by certain microorganismexhibited antimicrobial activity against important human pathogenicbacteria and fungi, as well as having antiviral and anti-tumouractivities (Zhao et al., 2006; Wachsman et al., 1999).

An object of the present invention is to provide a novel antitumouragent which has excellent antitumour activity based on metabolites withreduced side effects and which can be prepared starting with a highlysafe bacterium used in food production. Yet, another objective of thispresent invention provides methods of measuring anticancer efficacy(cancer-cell-growth inhibition and apoptosis induction) of Lactic acidbacteria, and further to provide screening method of the Lactic acidbacteria which have anticancer efficacy.

SUMMARY OF INVENTION

Accordingly, the present invention relates to the use of a Lactic acidbacteria strains to prevent or treat a tumour, wherein the bacteriastrains being Lactobacillus plantarum I-UL4, TL1, RS5, RG14, RG11 andRI11 deposited at the BIOTEC Culture Collection (BCC), BIOTEC CentralResearch Unit of Thailand (the bacteria strains are in a live form ornone live but intact). Moreover, the Lactobacillus plantarum I-UL4, TL1,RS5, RG14, RG11 and RI11 is said to produce metabolite(s) such asbacteriocins which is capable to inhibit proliferation and induceapoptosis of cancer in a mammal (preferably human being) in order toprevent or treat tumour. In particular, the metabolite is an anti-tumouragent and the metabolite(s) is supported with nutrients, vitamin(preferably vitamin B), salt of organic acids (preferably sodium salt offormic acid, acetic acid and lactic acid) or combination thereof.Indeed, the amount of metabolite(s) use in this particular invention isat a range between 0% (v/v) and 50% (v/v) of the total complete growthmedia. Following to this, it is said that the anti-tumour agent providesthe means of reducing viability in various types of human cancerouscells (e.g human breast cancer cell line MCF-7, human colorectal cancercell line HT-29, human cervical cancer cell line HeLa, human livercancer cell line Hep G2, human leukemia cell lines HL-60 and K-562) byproviding a concentration of 1% (v/v) to 50% (v/v). In addition, theanti-tumour agent provides the means of inhibiting the proliferation invarious types of human cancerous cells by having a concentration of 1%(v/v) to 50% (v/v). Also, the present invention illustrate Lactic acidbacteria strains having to facilitate induction of apoptosis of thecells of a cancer, wherein the strains being Lactobacillus plantarumI-UL4, TL1, RS5, RG14, RG11 and RI11 deposited at the BIOTEC CultureCollection (BCC), BIOTEC Central Research Unit of Thailand. Accordingly,the present invention discloses manufacturing of a drug destined for thetreatment or prevention of cancer, wherein the drug is manufactured bymeans of Lactobacillus plantarum I-UL4, TL1, RS5, RG14, RG11 and RI11strains. In fact, the anti-tumour agent also provides a combination of 6types of metabolites derived from Lactobacillus plantarum I-UL4, TL1,RS5, RG14, RG11 and RI11 deposited at the BIOTEC Culture Collection(BCC), BIOTEC Central Research Unit of Thailand.

Yet, another aspect of the present invention relates to composition totreat or prevent cancer, comprising an effective quantity of at leastone Lactic acid bacteria strains and a pharmaceutically acceptablevehicle, said strain(s) being Lactobacillus plantarum I-UL4, TL1, RS5,RG14, RG11 and RI11 is deposited at the BIOTEC Culture Collection (BCC),BIOTEC Central Research Unit of Thailand. It is said that, thecomposition contains an anti-tumour agent which include metabolites suchas bacteriocins and organic acids (includes sodium salt of formic acid,acetic acid and lactic acid) and further contains nutrients, vitamin(preferably vitamin B), and salt of organic acids or combinationthereof. Also, the composition further includes a combination of 6metabolites derived from Lactobacillus plantarum I-UL4, TL1, RS5, RG14,RG11 and RI11 strains deposited at the BIOTEC Culture Collection (BCC),BIOTEC Central Research Unit of Thailand.

In addition, the present invention also relates to a method tofacilitate apoptosis of cancer cells in a mammal (human being) and themethod comprises the administration the composition mentioned above. Inparticular, this invention preferably provides a kit for preventing ortreating a cancer in a mammal, wherein the kit comprises a containercontaining the composition as mentioned above. Also, the presentinvention discloses foodstuffs for anti-tumour containing Lactobacillusplantarum I-UL4, TL1, RS5, RG14, RG11 and RI11 strains deposited at theBIOTEC Culture Collection (BCC), BIOTEC Central Research Unit ofThailand. Also, the foodstuffs includes a combination of 6 metabolitesderived from Lactobacillus plantarum I-UL4, TL1, RS5, RG14, RG11 andRI11 strains.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be fully understood from the detaileddescription given herein below and the accompanying drawings which aregiven by way of illustration only, and thus are not limitative of thepresent invention, wherein:

FIG. 1 is a graph of anti-proliferative effect of metabolites derivedfrom the six strains of Lactic acid bacteria on MCF-7 cells. Valueswithin the same row and experiment having an asterisk are significantlydifferent from untreated control, P<0.05.

FIG. 2 is a graph of anti-proliferative effect of metabolites derivedfrom the six strains of Lactic acid bacteria on HT-29 cells. Valueswithin the same row and experiment having an asterisk are significantlydifferent from untreated control, P<0.05.

FIG. 3 is a graph of apoptosis induction of Lactic acid bacteriametabolites on MCF-7 cells. Notes: “early apop” indicated earlyapoptotic cells and “late apop” indicated late apoptotic cells andnecrotic cells. T1: untreated control; T2: treatment group (±indicatesstandard error). The treatment group is compared to untreated control.Values within the same row and experiment sharing a common superscriptletter are not significantly different, P>0.05.

FIG. 4 is a picture of fluorescence photomicrographs of MCF-7 cellstreated with UL4 metabolite. Panel A: 24 hours, B: 48 hours and C: 72hours. Morphological changes following exposure to Lactic acid bacteriametabolite are typical of apoptosis, showing ↑a=cell shrinkage,↑b=membrane blebbing, ↑c=apoptotic bodies formation, and ↑d, necroticcells. (Magnification: 400×).

FIG. 5 is a graph of two-parameter scatterplots (left panel) and singleparameter histogram (right panel) illustrating the detection of DNAstrand breaks in apoptotic cells by TUNEL assay. Panel A: 72 h untreatedcontrol; B: 72 h treated with UL4 metabolite. Apoptotic cells (R4) arecharacterized by very high frequency of DNA strand breaks (noteexponential scale of Y coordinate) in scatterplots and M2 gate inhistogram.

FIG. 6 is a sequence listing of Lactobacillus plantarum I-UL4, TL1, RS5,RG14, RG11 and RI11 strains deposited at the BIOTEC Culture Collection(BCC), BIOTEC Central Research Unit of Thailand

DETAILED DESCRIPTION OF THE INVENTION

In particular, the present invention describes the cytotoxic effects ofmetabolites of Lactic acid bacteria strains isolated from Malaysianfoods, Lactobacillus sp., on various cancerous cells without cytotoxiceffects on normal cells. The present invention also relates to theinduction of cell death on human cancerous cells. More particularly, thepresent invention relates to inhibition of proliferation of colon andbreast cancer cells. More specifically, the present invention relates tothe induction of apoptosis on human breast cancer cells.

It was demonstrated that the present invention provides a method foreasily measuring the antitumour effect of Lactic acid bacterium, amethod for screening a Lactic acid bacterium having an antitumour effectby using the method, a method for easily measuring an anti-inflammatorycytokine effect of Lactic acid bacterium and a method for screening aLactic acid bacterium having an inflammatory cytokine inhibitory effectby using the method.

The solution is based on that the present invention, relates to a novelLactobacillus genus microorganism and more particularly, Lactobacillusplantarum including I-UL4, TL1, RS5, RG14, RG11 and RI11 strainsisolated from food sources. In particular, these strains are said to behaving immune enhancement, anticancer activities and a use thereof. Dueto its excellent anticancer activities by reducing the cancer cellviability, inhibition on proliferation of cancer cells and induction ofapoptosis on human cancer cells, the Lactobacillus plantarum includingI-UL4, TL1, RS5, RG14, RG11 and RI11 strains of the present inventioncan be effectively used for the production of various products such asanticancer agent, food additive, feed additive, health supplement orfunctional food in nutraceutical industry and pharmaceutical products.

Accordingly, the I-UL4, TL1, RS5, RG14, RG11 and RI11 strains wereobtained from the Department of Bioprocess Technology, Universiti PutraMalaysia. Indeed, the strains are capable of producing metabolites. Uponobtaining the metabolites from these stains, the metabolites aretransferred in a fresh universal bottle and the pH of the metaboliteswere adjusted to a physiological pH using sodium hydroxide (NaOH).Moreover, the metabolites were filtered through 0.22 μm membrane syringefilter prior to treatment on cancer cells. Later, the metabolites werekept at 4° C. till use.

Without being limited to theory, it is believed that the metabolitesderived from probiotic Lactic acid bacteria are capable of significantlyreducing cancer cell viability, inhibiting proliferation of cancer cellsand inducing apoptosis on human cancer cells. See working examplesherein for further details.

BEST MODE TO CARRY OUT THE INVENTION

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims. When a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. When the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited. It must be noted that as used herein and in the appended claims,the singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

EXAMPLES

The following example serves to illustrate the scope of the use of thepresent invention and not to limit its scope. Modifications andvariations may be made to it without going away from neither the spiritnor the scope of the invention. Even though one may use other methods orproducts equivalent to those that we find hereinafter to test or tocarry out the present invention, the material and the preferred methodsare described. In the context of the present invention, in order todetermine how the Lactic acid bacteria help in the apoptosis of cancer,trials have been conducted on the human breast cancer cell line MCF-7,human colorectal cancer cell line HT-29, human cervical cancer cell lineHeLa, human liver cancer cell line Hep G2, human leukemia cell linesHL-60 and K-562. The Lactic acid bacteria used constitute ofLactobacillus plantarum including I-UL4, TL1, RS5, RG14, RG11 and RI11strains. Additionally, these strains produce metabolite/s which acts asan anti-tumour agent or cytotoxic agent. Yet, in this present invention,the anti-tumour agent or cytotoxic agent is supported by bacteriocins,nutrients, vitamin, salt of organic acids.

Preparing Metabolite

-   -   i) The metabolites of Lactobacillus sp. was transferred to fresh        universal bottle and the pH of the metabolites was adjusted to        physiological pH (between 7.2 and 7.4) using NaOH. The        metabolites was filtered through 0.22 μm membrane syringe filter        (Milipore, USA) prior to treatment on both cancerous cell lines        and normal cells.

Obtaining Cell Line

-   -   1) The Animal Tissue Culture Laboratory of Universiti Putra        Malaysia provided the human breast cancer cell line MCF-7, human        colorectal cancer cell line HT-29, human cervical cancer cell        line HeLa, human liver cancer cell line Hep G2, human leukemia        cell lines HL-60 and K-562. As for non-malignant normal        glandular epithelium cells MCF-10A which was used as a model of        normal glandular epithelium was obtained from the American Type        Culture Collection (ATCC). All cancer and normal cell lines were        maintained in the ATCC recommended medium supplemented with 10%        (v/v) heat-inactivated Foetal Bovine Serum and 100 U/ml        penicillin-streptomycin, and incubated at 37 ° C. in 5% CO₂        atmosphere.    -   ii) All animal experiments were conducted adhering to the        guidelines of Faculty of Veterinary and Animal Sciences,        Universiti Putra Malaysia. The 7-8 weeks old male ICR mice were        sacrificed by cervical dislocation. The spleen and thymus        obtained after dissection were rinsed 2-3 times with ice-cold        PBS followed by mincing on a wire mesh soaked in PBS. The cell        suspension was filtered using a wire mesh to remove any cell        clumps. All the cells obtained were washed 2-3 times with        ice-cold PBS by centrifugation (300×g for 5 min).        -   ii) With the guideline and consent obtained from Faculty of            Veterinary and Animal Sciences, Universiti Putra Malaysia,            about 10 ml of blood was drawn (venipuncture) aseptically            from healthy human volunteers (25-30 years of age) and            transferred to preservative free heparin tube.            Anticoagulated blood was diluted with equal volume of pH 7.5            phosphate buffered saline (PBS) and slowly layered over            Ficoll-Paque Plus. The mixture was centrifuged in a swinging            bucket rotor at 400×g for 40 min at 18-20° C. Peripheral            blood mononuclear cells (PBMC) were collected from            Ficoll-plasma interface and washed twice with PBS. The cells            pellets of mice splenocytes, thymocytes and human PBMC were            resuspended in complete growth media with density of 5×10⁵            cells/ml.

Measures of Viability of Cancer Cells, Proliferation and ApoptosisInduction

-   -   1) The effect of metabolites derived from six strains of locally        isolated Lactobacillus sp. on cell viability was assessed using        MTT assay according to Mosmann (1983). Cells were plated onto        flat-bottomed 96-well plates at the density of 1×10⁴ cells/well        for 24 h prior to treatment [control cells, 0% (v/v) of        metabolites] or in the presence of two-fold dilution of        concentrations [50% (v/v) to 0.5% (v/v)] of metabolites. After        24 h, 48 h, and 72 h of incubation respectively, 20 μl of MTT        solution (5 mg/ml in PBS) was added to each well and incubated        in the dark for 4 h at 37° C. and 5% CO₂ atmospheric condition.        The resultant formazan crystals were dissolved in 100 μl of        DMSO. The absorbance (A) at 570 nm with reference wavelength of        630 nm was then recorded usingμ Quant ELIZA reader (Biotek        EL340, USA). Percentage of cell viability is calculated as        (A_(sample)−A_(blank))/(A_(control)−A_(blank))×100%. All        experiments were conducted in three independent experiments with        triplicate samples for each experiment and the concentration of        50% inhibition (IC₅₀) values were determined.

TABLE 1 Concentration of metabolites derived from six strains ofLactobacillus sp. showing 50% inhibition (IC₅₀ values) on various cancercell lines and normal cells after 72 h of incubation. IC₅₀ values ofmetabolites derived from Lactobacillus sp. Type of cells UL4 TL1 RS5RG14 RG11 RI11 Cancer cell lines MCF-7 cells 10 13 21 20 16 16 HeLa 2018 24 20 N.D. 18 Hep G2 22 22 27 22 N.D. N.D. HT-29 N.D. N.D. 28 22 N.D.N.D. K-562 10 5 5 5 5 5 HL-60 5 5 9 10 1 1 Normal cells MCF-10A 26 N.D.N.D. N.D. N.D. N.D. Human PBMC N.D. N.D. N.D. N.D. N.D. N.D. Micesplenocytes N.D. N.D. N.D. N.D. N.D. N.D. Mice thymocytes N.D. N.D. N.D.N.D. N.D. N.D. Notes: IC₅₀ value, % (v/v) of metabolites causing 50%inhibition of cell viability of treated cells. N.D., no IC₅₀ value wasdetected up to concentration of 50% (v/v).

-   -   iii) Cells were seeded at 5×10³ cells/well in 96-well plate. The        medium was aspirated after 24 h and replaced with fresh medium        containing the test extract to be studied for 24 h, 48 h and 72        h, respectively. Further procedure was carried out according to        protocol from manufacturer. Using a BrdU ELISA system as an        alternative to the radioactive [³H] thymidine incorporation        assay, cells were reincubated with BrdU subsequently to a final        concentration of 10 μM for 2-24 h. Medium was aspirated from        each well. Fixative/denaturing solution was added to enable        antibody binding to the incorporated BrdU where cells were        fixed, permeabilized and the DNA denatured. Anti-BrdU antibody        was added to every well and incubated for 1 h to bind to        incorporated BrdU. Unbound antibody was then washed away and        horseradish peroxidase-conjugated goat anti-mouse which binds to        the detector antibody was added. Stop solution was added to each        well before the colored reaction product is quantified by        measuring the absorbance of each well at 450 nm (reference        wavelength 540 nm) using spectrophotometer (Biotek EL340, USA).    -   iv) One of the hallmarks of apoptotic cells is the        externalization of phosphatidylserine (PS). In principle,        annexin V detects cell surface phosphatidylserine while PI        stains cellular DNA of cells with compromised cell membranes.        This allows the discrimination of viable cells (annexin V⁻/PI⁻)        from early apoptotic cells (annexin V⁺/PI⁻) and late apoptotic        and necrotic cells (annexin V⁺/PI⁺). In this study, mode of cell        death was examined using the BD Biosciences Annexin V-FITC        Apoptosis Detection Kit. MCF-7 cells were seeded into six-well        tissue culture plates at a density of 2.5×10⁵ cells/well and        allowed to attach overnight, followed by treatment for 24, 48        and 72 h. At the end of treatment, detached and trypsinised        cells were pelleted down and resuspended in binding buffer.        Cells were then stained with annexin V-FITC and PI for 15 min in        the dark and diluted with binding buffer to a final volume of        500 μl before flow cytometry analysis. A total of 10,000 events        per sample were acquired (Vermes et al., 1995). Data acquisition        and analysis were perforated on FACS-Calibur flow cytometer (BD        Biosciences, USA). Results are expressed as the mean±SEM of at        least three separate experiments.    -   v) MCF-7 cells were treated with UL4 metabolite in six well        plates and were incubated in 5% CO₂ atmospheric condition at        37° C. for 24, 48 and 72 hours. After 24, 48 and hours of        incubation, respectively, detached cells in the medium were        collected and added back to trypsinised adherent cells. The        cells were washed with PBS and then incubated with 10 μl of AO        (100 μg/mL) and PI (100 μg/mL) at a ratio of 1:1 in 1 mL of        cells and recentrifuged at 200×g for 5 min. The supernatant was        aspirated, leaving 50 μl of remnant supernatant. The pellet was        resuspended and 10 μl of cell suspension was dripped on slide        and covered with cover slip. Within 30 min, the slide was        observed under fluorescent microscope (Nikon FC-35DX, Japan)        with combination of excitation filter and barrier filter of        450-490 nm and long pass filter of 520 nm. The percentages of        viable (green intact cells), apoptotic (green shrinking cells        with condensed or fragmented nucleus), and late apoptotic and        necrotic (red cells) were determined from >200 cells for the        data to be statistically significant.    -   vi) Cells treated with UL4 metabolite were harvested after        incubation period of 24, 48 and 72 hours. The detached and        trypsinised cells were pelleted and fixed with ice cold 80%        ethanol drop by drop and incubate at 4° C. until analysis        (minimum 2 hours). Then, the cell was pelleted again and washed        with PBS-BSA-Az-EDTA buffer for twice. The cells were        pulse-vortexed to elute the DNA fragments. For DNA analysis,        cells were stained in PBS buffer consists of 0.1% (v/v) Triton        X-100, 10 mM EDTA, 50 μg/mL RNase A and 2 μg/mL PI. This process        was carried out in the dark because PI is sensitive to light.        The cell was then incubated for 30 min at 4° C. and then read        with FACS-Calibur flow cytometer (BD Biosciences, USA) at Animal        Tissue Culture Laboratory, Department of Molecular Biology,        Faculty of Biotechnology and Biomolecular Sciences, University        Putra Malaysia within 24 hours. Ten thousand events per samples        were acquired. Doublets and cell debris were gated off in DNA        dot plot view before calculation. Cell cycle distribution was        calculated using CELLQuest Pro software (BD Biosciences, USA).        The experiment was repeated for at least three times with        triplicated samples for each experiment.

TABLE 2 Effect of UL4 metabolite on cell cycle phases distribution ofMCF-7 untreated UL4 control (%) metabolite (%) 24 hours Sub- G₀/G₁ 0.5 ±0.3 1.7 ± 1.6 G₀/G₁ 59.1 ± 3.1  64.5 ± 6.7  S 21.4 ± 4.2  16.4 ± 3.6  M19.3 ± 1.6  17.6 ± 3.3  48 hours Sub-G₀/G₁ 0.5 ± 0.2 14.3* ± 5.6   G₀/G₁80.1 ± 6.2  62.7 ± 2.1  S 9.4 ± 3.7 13.7 ± 2.7  M 10.1 ± 2.6  9.7 ± 1.172 hours Sub- G₀/G₁ 0.5 ± 0.1 9.5* ± 4.7  G₀/G₁ 89.9 ± 1.4  70.2 ± 3.4 S 3.3 ± 0.4 9.9 ± 1.1 M 6.2 ± 1.5 10.1 ± 1.2  Notes: The data shownabove the bars represent the mean of percentage of cells. Error barsrepresent SEM. Values within the same row and experiment having anasterick are significantly different (P > 0.05).

-   -   (vi) DNA labeling was carried out using BD Biosciences        APO-DIRECT Kit. Cells were seeded at 2×10⁵ cells/well in        six-well tissue culture plate. After 24 hours, the medium was        replaced with fresh medium containing the test extracts to be        studied at the desired concentrations. Further procedure was        done according to manufacturer's instructions. After incubation,        the detached and trypsinised cells were pelleted and resuspended        in 1% (w/v) paraformaldehyde in PBS (pH 7.4) at a concentration        of 1-2×10⁶ cells/mL. The cell suspension was placed on ice for        30-60 min. Fixed cells were then collected by centrifugation at        300×g for 5 min and the supernatant was discarded. The cell        pellets were washed twice in 5 mL of PBS and were resuspended in        the residual PBS in tubes by gently vortexing. Subsequently,        cells were resuspended in 70% (v/v) ice-cold ethanol and left        for a minimum of 30 min on ice before being stored at −20° C.        After appropriate storage time (1-7 days), the cell suspension        was centrifuged at 300×g for 5 min and the 70% v/v ethanol was        aspirated. The cells were washed with 1 mL of Wash Buffer twice        and the cell pellet was resuspended in 50 μl of the DNA Labeling        Solution.    -    After incubation of 60 min, the cells were rinsed with 1 mL of        Rinse Buffer twice and pelleted by centrifugation at 300×g for 5        min. The cell pellet was stained with 0.5 mL of the PI/RNase        Staining Buffer. The cells were incubated in dark for 30 min at        RT and analyzed by FACS-Calibur flow cytometry using CELLQuest        Pro software within 3 hours. Ten thousand events per sample were        acquired with the cell doublets and debris were gated off in DNA        dot plot view before calculation.    -    Results were expressed as mean ±S.E and analyzed by General        Linear Model. The statistical analysis was conducted using        Minitab Statistical Software at differences of P <0.05.

We claim:
 1. A composition for treating or preventing cancer, whereinsaid composition comprises an effective quantity of at least one lacticacid bacteria strain, and/or a metabolite from said bacteria strain, anda pharmaceutically acceptable vehicle, wherein said bacteria strain isselected from the group consisting of Lactobacillus plantarum I-UL4,TL1, RS5, RG14, RG11 and RI11 deposited at the BIOTEC Culture Collection(BCC), BIOTEC Central Research Unit of Thailand.
 2. The compositionaccording to claim 1, characterized in that said bacteria strain is in alive form or a non-live, but intact, form.
 3. The composition accordingto claim 1, wherein the composition comprises a metabolite of saidbacteria strain.
 4. The composition according to claim 1, wherein thecomposition further comprises a nutrient, vitamin, or a salt of anorganic acid, or a combination thereof.
 5. The composition according toclaim 4, wherein the vitamin is vitamin B.
 6. The composition accordingto claim 4, wherein the salt of an organic acid is a sodium salt offormic acid, acetic acid or lactic acid.
 7. The composition according toclaim 1, wherein the composition comprises a combination of 6metabolites derived from Lactobacillus plantarum I-UL4, TL1, RS5, RG14,RG11 and RI11 strains deposited at the BIOTEC Culture Collection (BCC),BIOTEC Central Research Unit of Thailand.
 8. A kit for preventing ortreating a cancer in a mammal, wherein the kit comprises a containerhaving enclosed therein a composition as defined in claim
 1. 9. Ananti-tumour foodstuff comprising a bacteria strain, and/or a metabolitefrom said bacteria strain, wherein said bacteria strain is selected fromthe group consisting of Lactobacillus plantarum I-UL4, TL1, RS5, RG14,RG11 and RI11 strains deposited at the BIOTEC Culture Collection (BCC),BIOTEC Central Research Unit of Thailand.
 10. The foodstuff according toclaim 9, wherein the foodstuff includes a combination of 6 metabolitesderived from Lactobacillus plantarum I-UL4, TL1, RS5, RG14, RG11 andRI11 strains deposited at the BIOTEC Culture Collection (BCC), BIOTECCentral Research Unit of Thailand.